PROJECT SUMMARY This grant application responds to Program Announcement Number PA-18-398 and outlines a training and research program which will prepare me for a long-term research career focused on the development of optical imaging methods to better understand, diagnose, and treat human prostate cancer. The proposal outlines a mentored-training program which places me under the direct guidance of a team of NIH funded researchers from the University of Washington (UW) and the neighboring Fred Hutchinson Cancer Research Center (FHCRC). My training plan involves three complimentary areas of focus: (1) receiving further training in tissue clearing and labeling, (2) obtaining educational and hands-on training in single-cell isolation and analysis, and (3) developing a knowledge-base in molecular assays and prostate cancer to facilitate meaningful future collaborations with experts in these fields. To achieve these goals, I will use the funding to dedicate time for attending graduate courses, workshops, training seminars, grand rounds lectures, and most importantly regular meetings with my entire career award team, as outlined in my career development plan. The research project component of the proposal is driven by the fact that rare cell populations are known to play an outsized role in many of the most challenging problems in cancer, and downstream profiling (i.e., genomics, proteomics, and transcriptomics) of these individual cells can therefore allow for more-effective therapies targeted against these often-hidden lynchpins of the disease. Despite this, single-cell isolation (the process by which individual cells are targeted and collected for further study) is still technically challenging with current methods, particularly for rare tumor cells. This is particularly problematic, as these rare tumor cells often dictate metastasis, treatment resistance, and recurrence, all of which ultimately result in patient mortality. While this challenge exists for nearly all forms of cancer, this proposal will focus specifically on metastatic tumor cells which have invaded the lympho-vascular space in prostate cancer, as the isolation and analysis of these rare cells can enable the development of new diagnostic and treatment strategies. To achieve this, I propose to build upon my post- doctoral research, and with the necessary training above, develop a novel light-sheet micro-aspiration microscope which will enable the isolation of these rare tumor cells from intact clinical specimens for the first time. The project is comprised of three specific aims: (1) development of a multi-resolution open-top light-sheet microscope system for identifying rare tumor cells; (2) combined demonstration of light-sheet microscopy with micro-aspiration for isolating rare tumor cells; and (3) proof-of-concept clinical validation of the system for isolating rare invading tumor cells from human prostate tissues with lympho-vascular invasion. The prostate cancer research environment at UW and the FHCRC is an ideal place for me to train and pursue this research project, and with the guidance of my mentorship team, I will successfully complete this career development award and pursue my long-term career goal of becoming an independent investigator. !